ACE gene polymorphism and susceptibility to hypertension in a Jordanian adult population

Hypertension is one of the most common and complicated disorders associated with genetic and environmental risk factors. The angiotensin-converting enzyme (ACE) is important in the renin-angiotensin-system pathway. The gene expression of ACE has been investigated as a possible hypertension marker. This study investigates the association between polymorphisms within the ACE1 and ACE2 genes and hypertension susceptibility in a Jordanian population. The study comprised a total of 200 hypertensive patients and 180 healthy controls. A polymerase chain reaction (PCR) was performed to genotype the candidate polymorphism (rs4646994) of the ACE1gene. The Luminex DNA array technique was used for genotyping SNPs (rs4359, rs4344, rs4341, rs4343, and rs2106809) of the ACE1 and ACE2 genes. Our findings suggest no association between SNPs and hypertension regarding allelic and genotypic frequencies. However, rs4359 was significantly associated with diet (pP = 0.049), know HTN (P = 0.042), and number of years DM (P = 0.003). rs4341 was associated with diet (P = 0.032), peripheral vascular disease (P = 0.005), and chronic kidney disease (p = 0.049). While rs4343 was associated with diet (P = 0.031), diabetes mellitus (P = 0.032), and other medication (P = 0.025). Furthermore, the haplotypes of four SNPs of the ACE1 gene showed no significant association with HTN patients and healthy controls. Our findings indicate no association between the polymorphisms in the ACE gene and the risk of hypertension development in the Jordanian adult population.


Introduction
Hypertension (HTN) is characterized by elevated systolic and diastolic blood pressure [1].It appears to result from a complicated interplay between genetic and environmental factors.Furthermore, it is a significant hazard factor for mortality and disability worldwide [2,3].It is vastly associated with cardiovascular diseases (CVDs).In addition, it is implicated in substantially increasing the risk of stroke, cerebrovascular diseases, end-stage renal disease, organ damage, and other medical disorders [4][5][6][7].
The global prevalence of HTN was estimated to be 972 million in 2000.And it significantly rose to 1.3 billion in 2015 [8].In Arab countries, it was estimated that 30 percent of adults suffered from HTN. [9].Also, among Jordanians, one-third of adults suffer from HTN [10].The risk factors that raise the risk of developing HTN can be divided into those that are modifiable, which are influenced by smoking, obesity, an excessive amount of salt, excessive alcohol consumption, and chronic stress.As well as non-modifiable ones, such as older age and family history [11].Besides that, genetic risk factors account for approximately 30-50 percent of blood pressure variation [12].
Multiple studies have revealed that the renin-angiotensin system (RAS) has substantial direct participation in the regulation of blood pressure and is composed of several genes, such as renin, angiotensinogen (AGT), angiotensin II type 1 receptor (AGTR1), and angiotensin-1-converting enzyme (ACE) [13,14].Angiotensin I-converting enzyme (ACE) is the main factor regulating blood pressure [15].Also, it regulates the renin-angiotensin system (RAS) and the Kinin-Kallikrein system [16,17].The ACE catalyzes the production of the vasoactive octapeptide (Ang II).Moreover, it inactivates the vasodilator bradykinin [18].
The human ACE gene spans approximately 21 kb and comprises 26 exons and 25 introns.It is located at location 23.3 on the long arm of chromosome 17 [19].A specific variation in the ACE gene is called the ACE I/D polymorphism, which is determined by the 287 bp Alu repeat sequence's presence (insertion) (I allele) or absence (deletion) (D allele) on ACE gene's intron 16 [20].In 1990, Rigat B et al. discovered the ACE gene polymorphism for the first time, and I/ D polymorphism's physiological significance was found through its association with plasma ACE levels, frequently studied in cardiovascular, hypertension, and other complex diseases [21].
Angiotensin II-converting enzyme (ACE2) was identified as the first reported ACE homolog in 2000 [22].It promotes and induces vasodilation via the effective degradation of Ang II and the production of the vasodilator Angiotensin Ang 1-7 [23,24].ACE2 negatively regulates the renin-angiotensin system [33]-also the alternate arm of the RAS [25].The ACE2 gene in humans is present along the short arm of Chromosome X, specifically position Xp22.2,spans approximately 39.98 kb of genomic DNA and contains 20 introns and 18 exons [26].
ACE1 and its homolog ACE2 are thus seen as effector regulators of blood pressure that have a counterbalancing role via the synthesis of vasoactive peptides in the renin-angiotensin system [27,28].Meanwhile, disturbance of the tissue ACE/ACE2 homeostasis may result from functional ACE/ACE2 gene polymorphisms.Therefore, it could cause variations in blood pressure.Elevated blood pressure results from ACE2 deficiency, whereas higher ACE2 expression safeguards against elevated blood pressure [29].Various previous studies have demonstrated and provided evidence that genetic variation in ACE genes (i.e., ACE I/D (rs4646994), G2350A (rs4343), (rs4341), (rs4344), (rs4359), (rs2106809)) is correlated with the pathogenesis of HTN [30][31][32][33][34]. Numerous gene families have been implicated in cardiovascular diseases, with multiple genetic variations spread across the Jordanian population.Research has also indicated that several SNPs may also influence the response to cardiovascular medications such as warfarin [35][36][37][38][39][40][41][42][43][44].However, the relationship between polymorphism within the ACE gene and HTN is still debatable and has so far been inconclusive; the reason is ethnic variations and geographic features.
The study investigated the association between single nucleotide polymorphisms within the ACE1 and ACE2 genes and HTN susceptibility.Furthermore, it represents the first investigation into the association of rs4646994, rs4359, rs4344, rs4341, rs4343 in ACE1, and rs2106809 in ACE2 gene with HTN risk in a Jordanian population.

Study subject
The patient cohort analyzed in our study comprised individuals who regularly visit hypertension clinics within the Coronary Cardiac Care Department at King Abdullah University Hospital (KAUH) in Irbid, Jordan.The diagnosis of hypertension relied on the ICD-10 coding system, thus ensuring the inclusion of patients with confirmed hypertension.Given the regular attendance of these individuals at hypertension clinics and their continuous treatment regimen, further verification of the disease was considered unnecessary.Thus, our study collected blood samples and clinical records using the specified methodology.Patients enrolled in the case-control study were recruited from a single institutional hospital, and the sample size was restricted to the number of patients visiting the outpatient cardiac clinic and the Coronary Heart Care Department and recorded in the hospital.
In our case-control study, only 200 unrelated Jordanian patients were included after excluding patients who did not meet the inclusion criteria.The inclusion criteria necessitated official confirmation of hypertension diagnosis, data availability in the KAUH patient registry system, and participants aged 35 years or older.The patients who received various antihypertensive drugs were recruited from the outpatients of the cardiac clinic and the Coronary Heart Care Department during their follow-up visits at King Abdullah University Hospital (KAUH) and were classified into five groups based on the type of antihypertensive medication they were prescribed (ACEIs, ARBs, CCBs, BBs, and TDs), following the guidelines determined by the American Heart Association (AHA) and the Joint National Committee on the Detection, Evaluation, and Treatment of High Blood Pressure (JNC), as practiced at KAUH.The exclusion criteria included individuals who refused to provide written informed consent, those with insufficient clinical data, and individuals who were related up to the second degree with participants in the current study.
In addition, 180 unrelated adult healthy volunteers with no history of HTN were randomly chosen from the Jordanian population as controls.All research volunteers provided written informed consent.Ethical approval was received from the Institutional Review Board (IRB) committee at the Jordan University of Science and Technology with attached code No (4/133/ 2020) informed consent.All patients' information was collected and analyzed, including patients' medical records data, along with data on demographics (age, height, weight, and gender) and lifestyle (smoking status and diet).
According to the WHO, 1.3 million adults in Jordan between the ages of 30 and 79 have hypertension, with a prevalence of 12%, out of the country's 10,699,000 total population (https://www.who.int/publications/m/item/hypertension-jor-2023-country-profile).The sample size was determined using the OpenEpi program, version 3.01, with a 95% confidence interval.Considering a prevalence of 12% of hypertension in Jordan, a precision of 5%, and a design effect 1, the sample size was calculated to be 163 patients.In our study, the sample size for the cases comprised 200 individuals.Initially, 320 patients were screened, but following the exclusion criteria, this number was reduced to 200, exceeding the requisite sample size.

General characteristics of HTN patients and healthy controls
In our study, 200 unrelated Jordanian patients were diagnosed with HTN and treated with various HTN medications.These patients included both males and females and the mean age ± SD was (58.84±10.39)years, with a range of  years.The control group had a mean age of (34.50±12.44),and the range was (18-80) years.The matched gender patients were selected with (58.4%) males and (41.6%) females, while the matched gender healthy controls were selected with (61.7%) males and (38.3%) females.Furthermore, all participants who met the inclusion criteria agreed and signed on to participate in our study.The patient's and healthy control descriptions, including demographic data, are shown in Table 1.

The candidate Gene and SNP selection
In our study, we selected six SNPs within candidate genes ACE1 and ACE2 that are specifically involved in susceptibility to HTN.The SNP database of the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/SNP/) and the Ensemble database (http:// www.ensembl.org/index.html) are two of the public databases from which these SNPs were selected.These SNPs were chosen based on their significant functional relevance, biological significance, and location within the selected gene.Furthermore, they are involved in the pathogenesis of HTN.
The results derived from RegulomeDB showed that ACE rs4359 was graded 1F with a score of 0.55436, rs4344 was graded 1b with a score of 0.36991, rs4341 was graded 4 with a score of 0.60906, rs4343 was graded 2b with a score of 0.57391, and rs2106809 was graded 7 with a score of 0.51392, indicating that SNPs were likely to affect binding and be linked to the expression of a gene target.In addition, the functional annotation of HaploReg v4.2 showed that rs4359, rs4344, rs4341, rs4646994, and rs2106809, located in the intronic region of the ACE1 gene and ACE2, among them, rs4343 was a synonymous coding SNP, potentially affecting both promoter and enhancer histone marks and changing the transcription factor binding motifs.Consequently, these markers could indirectly contribute to the pathogenesis of hypertension by modulating the corresponding transcription factors of ACE.

DNA preparation
The QIAGEN Puregene1 Blood Core kit-B extracted genomic DNA from frozen venous blood specimens according to the manufacturer's instructions.

DNA analysis and genotyping
The Nano-Drop ND-1000 ultraviolet-visible spectrophotometer (Bio Drop, UK) was used to check the quality and quantity of isolated DNA.The polymerase chain reaction (PCR) method was applied for the analysis of the I/D polymorphism (rs4646994) of the ACE1 gene using the primers flanking [38].Gene amplification was also carried out using a PCR assay.The mixture was heated under the following conditions: initial denaturation at 94C˚for 5 minutes, followed by 35 cycles of denaturation at 94C˚for 30 seconds, annealing at 58 C˚for 45 seconds, extension at 72C˚for 30 seconds , and final extension at 72C ˚for 2 minutes.
PCR products were visualized and separated by 3% agarose gel electrophoresis with ethidium bromide.The existence of 190 and 490 bp fragments allowed the identification of D or I alleles.Heterozygous D/I genotype show the existence of two bands at 190 bp and 490 bp.The Luminex DNA array technique was used for genotyping SNPs (rs4359, rs4344, rs4341, rs4343, Primers used in the study were chosen based on previously published scientific papers and derived from known human sequences.These are briefly summarized in Table 2.

Gel electrophoresis and genotyping of ACE1 gene
The genotypes of the SNP (rs4646994) in intron16 of the ACE1 gene were analyzed using polymerase chain reaction (PCR) and gel electrophoresis, and the resulting genotypes were visualized on an agarose gel.In terms of band sizes, three genotypes were detected: a homozygous (D/D) genotype at 190 bp and a homozygous (I/I) genotype at 490 bp, with the presence of heterozygous alleles (D/I) indicated by the appearance of both bands 190 and 490 bp.

Statistical analysis
The data analysis used the Statistical Package for the Social Sciences (SPSS) version 25.0 (SPSS, Inc., Chicago, IL, USA).Pearson's χ 2 -test and ANOVA were employed to perform genotypephenotype analysis, assessing the normal distribution of variables, which were presented as Mean ± SD and statistically tested.Additionally, SNPStats, a web-based tool (https://www.snpstats.net/start.htm), was utilized to analyze single SNPs, including examination under multiple inheritance models and assess Hardy-Weinberg Equilibrium.Furthermore, SNPStats facilitated the analysis of various SNPs, allowing for the estimation of haplotype frequencies and the investigation of associations between haplotypes and disease conditions.The risk associated with genotypes/alleles was evaluated by calculating the Odds Ratio (OR) with a 95% confidence interval (CI).Statistical significance was determined at p<0.05.In examining genotype-to-genotype correlations, we utilized a post hoc multiple comparison test.Furthermore, multinomial logistic regression analysis was used to evaluate the potential influence of gene polymorphisms on the disease.We determined adequate SNPs using the reference [45] method for multiple testing corrections.The significance cut-off was also set at α/n, where α = 0.05 and n represents the number of tests, employing the Bonferroni correction [46].

HWE test
All investigated SNPs met the criteria for HWE in both cases, and the controls were included in the study.One SNP (rs2106809) exhibited deviation from Hardy-Weinberg equilibrium (HWE) in both the control and patient groups, necessitating its exclusion from the analysis.In addition, SNP (rs4646994) demonstrated a deviation from HWE solely within the patient group.Notable, when the population deviates from the Hardy-Weinberg equilibrium, it means that particular mechanisms or processes drive evolution.Some variables might produce departures from HWE and upset the normal distribution, such as mutations, natural selection, gene flow, or the higher possibility of this divergence in our study, possibly due to non-random mating.Table 3 displays the candidate genes with their associated polymorphisms.The distribution of each SNP's minor allele in the cases and controls demonstrates standards-Weinberg equilibrium.

Association of SNPs candidate genes with HTN
In our study, the association between HTN and candidate polymorphisms was investigated.No significant associations were found for all SNPs in the ACE1 gene, and neither the allelic nor genotypic frequencies between cases and controls showed any significant differences.As shown in Tables 4 and 5.
This study used different genetic models in the genetic association analysis.Table 6 summarizes the genetic models (co-dominant, dominant, recessive, and over-dominant) and shows the chi-squared values between cases and healthy controls.
For the SNPs (rs4359, rs4344, rs4343, and rs4341) in the ACE1 gene, no genetic associations were found within all genetic models (co-dominant, dominant, recessive, and over-dominant) with HTN risk.The haplotype was studied as a part of the genetic association analysis; our findings revealed the haplotypes of four SNPs on the promoter of the ACE1 gene showed no significant association with HTN patients and healthy controls.As shown in Table 7.

Post hoc and regression analysis
In this research, regression analysis was utilized as a powerful stay robust method to explore the complex relationships between essential variables observed in the case and control groups.The Regression analysis sought to clarify the relationship between hypertension, symbolized by systolic and diastolic blood pressure measurements, and different clinical characteristics among the cases.By examining these relationships, we sought to identify possible factors influencing the onset and development of hypertension in our study sample.Moreover, our analysis expanded beyond simply exploration of the association between hypertension and clinical features.We also sought to clarify any possible relationship between particular genetic variants represented by the selected SNPs and the identified clinical features within the cases.This portion of the analysis allowed us to delve into the genetic foundations of hypertension and to identify any potential genetic markers related to particular clinical features of the condition.The comprehensive results of the regression analysis, which include the intricate relationships among hypertension, clinical features, and genetic variations, are presented in S2, S3 and S4 Tables.These tables present a comprehensive analysis of the statistical results and give essential details about the strength and direction of the correlations observed among the study cohort.Furthermore, to further explain the importance of our results, we performed post hoc analyses to compare individuals with different genotypes regarding clinical symptoms within

Discussion
Hypertension has been identified as a significant public health concern in the Middle East over several decades.Nevertheless, its impact exhibits pronounced heterogeneity, resulting in a widening health disparity between high-income and low to middle-income countries regarding various aspects, including awareness, diagnosis, treatment, and control [8].The Middle East, a region known for its diversity and primarily situated in West Asia, exemplifies these discrepancies, with the Gulf countries predominantly constituting high-income nations.In contrast, others fall within the spectrum of low to middle-income status [47].Moreover, this region is imbued with notable social, religious, and political variances, engendering substantial inequalities concerning access to healthcare, gender dynamics, daily lifestyles, and occupational opportunities [48].Recent years have witnessed a swift urbanization process in the Middle East, concurrent with a heightened burden of chronic diseases, amidst national preventive health systems that have lagged mainly behind [49].Given its relative ease of diagnosis, treatment, and management, hypertension presents a crucial focal point for scalable public health interventions in the region [50].
The prevalence of hypertension in the Middle East varies among countries and population groups.According to data from the Global Burden of Disease Study and other sources, hypertension prevalence in the Middle East has been steadily increasing.Based on a meta-analysis study in the Middle East, the overall prevalence of hypertension in the region was 24.36%.An increasing trend in the prevalence of hypertension was observed with increasing age [51].Studies conducted in various Middle Eastern countries have reported high prevalence rates of hypertension among adults.In Saudi Arabia, for instance, hypertension has been found to affect between 26% to 30% of the adult population [52].Similarly, research in Iran indicated a hypertension prevalence of approximately 25% among Iranian adults [53].Moreover, studies in Jordan have reported that the age-standardized prevalence was 33.8% among men and 29.4% among women aged 18 to 90. [54].
HTN is a global health issue affecting over one billion adults and causing more than 9.4 million mortalities annually [55].Only one in every four adults (24%) has HTN under control [56].Despite the availability of numerous classes of effective drug options, the control rate remains uncontrolled and far from optimal [57].It has been proven that the variants in the ACE genes affect the ACE plasma level and blood pressure regulation.Half, or 47%, of the overall phenotypic diversity of serum ACE levels is attributed to the insertion/deletion polymorphism [58].The (I/D) polymorphism directly correlates to the diversity in plasma ACE levels; the (I) allele is related to diminished tissue concentrations of angiotensin II and a diminished enzyme level.
Meanwhile, D alleles have a high ACE plasma level, increased concentrations of angiotensin II, and degradation of bradykinin, which leads to hypertension [59][60][61].Otherwise, in the linkage study, Ljungberg B et al., did not support that the ACE level is associated with HTN in both men and women, and there was no correlation between the ACE level and the systolic, diastolic or pulse pressure, which was not in agreement with most of the researchers [62].Previous studies have been conducted in large populations to investigate the pivotal role of the angiotensin-converting enzyme ACE I/D polymorphism in hypertension in an Indian, a Sikh, and an African American population [63][64][65].In contrast, our findings revealed that none of the SNPs in the ACE1 gene were linked to HTN in all genetic models.as shown in Tables 4-6.
In addition, the haplotype analysis revealed that the haplotypes of the ACE1 gene were not significantly associated with a risk of HTN in the Jordanian population.as shown in Table 7.
Following the HWE test for the investigated polymorphisms in the case and control sample sets, the genetic analysis for this study included all of the polymorphisms that satisfied the HWE criteria except for SNP (rs4646994) in the ACE1 in the case group and (rs2106809) in the ACE2.For that, any p-value less than (0.05) is considered not normally distributed, which would be for various reasons; the most likely is that the population size was not very large, making genetic drift negligible.Hardy-Weinberg equilibrium (HWE) tests are often used to rapidly examine genotype information, assuming that genotype frequencies ought to adhere to HWE proportions in a large population with random mating.Deviations from HWE, on the other hand, might result from a variety of different variables, such as purifying selection, copy number variation, inbreeding, or population-specific factors [66]; another possibility is that the Arabic population "including the Jordanian population," tends to marry among relatives.Relative marriages have a significant influence on the distribution of genotypes in a population and have the potential to disturb HWE.Under normal circumstances, HWE assumes random mating in which individuals pick their spouses without regard for bias.However, when relatives marry or engage in consanguineous relationships, "non-random mating, "the genetic diversity within the population becomes restricted, resulting in a higher probability of shared alleles and observing that homozygous alleles are much more prevalent in that population [67].
When considering why one SNP might deviate from HWE from others in the same sample, there are several possibilities, including Random fluctuations in allele frequencies that can occur in small populations due to genetic drift.If a particular SNP is in a genomic region with low genetic diversity or subject to founder effects, it is more likely to deviate from equilibrium [68].Natural selection can favor or reject specific alleles, causing deviations from HWE.If an SNP is under selection pressure due to its association with a beneficial or harmful trait, it may deviate from equilibrium [69].New mutations can introduce novel alleles into a population.If an SNP experiences a recent mutation event or is in a region with a high mutation rate [70], it may deviate from HWE until the population reaches a new equilibrium.Deviations from HWE may also occur due to linkage disequilibrium, where a SNP is physically close to other genetic variants that influence their allele frequencies [71].In such cases, deviations from equilibrium may be specific to certain SNPs due to their linkage with other genetic loci.These factors may explain the deviations from Hardy-Weinberg Equilibrium (HWE) for the particular SNP (rs2106809).To determine the type of deviations in rs2106809 SNP, we compare the observed genotype frequencies (AA, GA, GG) to the expected frequencies in the HWE equation (p 2 + 2pq + q 2 = 1).Based on this comparison, we observe an excess of individuals with the AA and GG genotypes compared to what is expected under HWE.In contrast, the observed frequencies of the GA genotype are lower than expected.The deviation observed in the genotype frequencies of the SNP (rs2106809) can be characterized as an excess of homozygotes (AA and GG genotypes) and a deficit of heterozygotes (GA genotype).
Additionally, the polymorphisms of the ACE2 genes have been proven to affect and alter gene expression and are linked to the risk of disorders like left ventricular hypertrophy, dyslipidemia, and hypertension [72,73].ACE2 polymorphisms have been identified as being correlated with hypertension, which is regarded as a prominent predictor of the severity of COVID-19 infection [74].Blackwood EM, et al., in their study, clearly showed that the ACE2 rs2106809 T allele might be functional, down-regulating ACE2 expression and reducing promoter activity in the absence or presence of other SNPs, resulting in a decrease in the expression rate of circulating angiotensin (1-7) and contributing to an increase in blood pressure and an increased HTN susceptibility [75].
Our study observed that rs4343 in the ACE1 gene was not associated with HTN risk among Jordanians.And there are no significant differences in the allelic and genotypic frequencies between cases and controls.These results support the idea that the rs4343 SNP in the ACE1 gene would not contribute to HTN development among the Jordanian population.These findings agree with the results of other studies conducted with different populations.These results were consistent with an exciting study by Wang et al., who clearly showed no significant association with HTN risk in a total of 400 HTN patients compared with 100 healthy controls in patients in the Hefei Region, Anhui, China [76].Besides that, these results agree with another study conducted in the Chinese Han population [77].
Conversely, however, specific studies of HTN in different populations find an association between the rs4343 polymorphism of ACE1 and HTN.In the Chinese Yi ethnic group, Yang et al. conducted a study using 244 HTN patients compared with 185 healthy controls and found an association between the rs4343 polymorphism of ACE1 and HTN susceptibility [78].Similarly, in the Emirati population, the rs4343 polymorphism has been associated with an increased risk of essential HTN [79].According to research by Jeong et al. on adult Koreans, the A allele of rs4343 raised the risk of hypertension by more than 2.1 fold, and high sodium intake enhanced this risk [80].Comparably, Wang et al. found that rs4343 was a risk factor for high pulse pressure levels linked to hypertension and arterial elasticity [81].
The current study reported no significant association between rs4341 and rs4344 polymorphism and the risk of developing HTN.In contrast, a study in a Mexican population reported a significant association of rs4344 with the risk of developing HTN [82].Meanwhile, rs4341 was associated with hypertension risk in the Pakistani population [83].Limited studies have investigated the association of ACE polymorphism, rs4341, and rs4344 with HTN.The results of the current study need further investigation on a large sample group.

Conclusions
This study is the first to find the association of single nucleotide polymorphisms (SNPs) of the angiotensin-converting enzyme (ACE) genes with HTN risk in Jordan.It confirms and provides evidence that the ACE2 gene has no essential role in HTN susceptibility in the Jordanian population.This study further supports the gene-environment interaction model of HTN.Given the limited research on pharmacogenetics and personalized medicine in Jordan [84,85], this study requires further investigations into other Arab populations with many samples to support and confirm our findings.

Table 1 . Description of the demographic characteristics of HTN patients and healthy controls.
b Mean ± Standard Deviation.

Table 5 . Genotype frequency of genes SNPs in HTN patients and controls.
these rigorous analytical techniques, we attempt to thoroughly investigate hypertension's complex, diverse nature, illuminating the complex interactions that arise between clinical features, genetic factors, and the development of this standard and intricate cardiovascular disease.